Purpose:
There is currently no conjunctival melanoma model available, limiting the ability to study the mechanisms of tumor progression which would allow the development of more effective chemotherapy and/or novel therapies, such as immunotherapy.

Methods:
In order to develop a xenogeneic model of human conjunctival melanoma in mice, cell lines derived from human conjunctival melanoma (CRMM-1, CRMM-2, CM2005.1) were injected orthotopically (4 x 106 cells/5 μl) into the subconjunctival space of immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Sequential passage of in vivo grown conjunctival melanomas was achieved by harvesting primary tumors, digestion of tumor tissue, and injection of tumor cells into the subconjunctival space of a new naive NSG mouse. Primary tumor growth was assessed by slit lamp examination, H&E, and immunohistochemical staining of conjunctival melanoma markers. Metastatic tumors derived from the cervical lymph nodes were also analyzed.

Results:
Xenogeneic human conjunctival melanomas grew progressively within the subconjunctival space of immunodeficient NSG mice. Primary tumors formed within two weeks in all mice (total of 101 mice; CRMM-1 n =33, CRMM-2 n = 34, CM2005.1 n = 35). All three cell lines expressed HMB-45. CRMM-1 and CM2005.1 expressed Melan-A, while S100 was only expressed at high levels in CM2005.1. On the primary tumors, the melanocyte markers..... were expressed. Histological analysis demonstrated all three cell lines had comparable growth and morphological characteristics: tumors grew along the subconjunctival space and sclera, displayed an epithelioid cell morphology with large nuclei, prominent nucleoli, and large cytoplasmic compartments with vacuoles. Surprisingly, initial primary tumors failed to metastasize in any mice, even after tumors attained the size requiring euthanasia. However, serial in vivo passage of primary tumors resulted in consistent metastatic tumor spread to the cervical lymph nodes draining the tumor-containing eye.

Conclusions:
Preventing metastatic spread of conjunctival melanoma is critical for a positive prognosis. Human xenogeneic conjunctival melanomas growing in immunodeficient NSG mice display tumor growth and metastatic progression that is similar to the human disease, allowing for the analysis of the protective effectiveness of current and novel therapies.